K+ fluxes, required for membrane potential maintenance and action potential shaping in excitable cells (e.g. neurons), are regulated by gating processes of voltage-gated K+ (Kv) channels. The activation gate (A-gate) opens by depolarization of the membrane rendering the channels conductive whereas closing the slow-inactivation gate (P-gate) leads to a long-lasting non-conductive state, thereby limiting the K+ currents available for membrane potential control. We showed recently that the A-gate, located at the cytosolic entrance of the pore and the P-gate, located at the extracellular opening of the channel, are coupled. The research proposal aims to determine the pathway of the coupling between the two gates. This includes the delineation of the role of cavity electrostatics in the coupling and the determination of nature of the motion of the S6 helix as a rigid body. In addition, we plan to discover alternative networks of interacting residues near the P-gate, which may result in non-cooperative movements of amino acid side controlling inactivation. We also hypothesize that the A- and P-gates are bi-directionally coupled, i.e., the status of the A-gate should influence the movement of the slow-inactivation P-gate. The proposed study may lead to fundamental results concerning the physiological functioning of voltage-gated ion channels including the communication between the activation and inactivation gates and the pathway for recovery form inactivation of the channels.

Using state-dependent cysteine accessibility we have studied the the molecular motions of the pore-lining S6 segments of voltage-gated Shaker K+ channels during inactivation gating. Based on the state-dependent modification pattern (MTSET/MTSEA) of cysteines engineered in S6 we concluded that S6 rotates around its own axis during inactivation gating. This movement of S6 may participate in the bi-directional coupling of the activation and inactivation gates of Shaker K+ channels. Using similar experimental technique, we precisely determined that the transitions among the 4 possible combinations of the activation and inactivation gate states 3 transitions are uni-directional: the channels locked in the open-inactivated configuration (OI) cannot recover from inactivation and closed (C) channels do not inactivate directly to the closed-inactivated (CI) state without visiting the open (O) state. Based on our experiments the gating scheme of Shaker K+ channels is CO->OI->CI->C. Using heavy water (D2O) we proved the functional existence of the H-bond network regulating inactivation kinetics, which was proposed earlier based on molecular modelling and X-ray crystallography.
In summary, our results contributed significantly to the understanding of the molecular mechanism of slow inactivation od Shaker K+ channels and the communication between the gates. In addition, we have established a new experimental technique in the laboratory which is unique in Hungary.